1. Field of the Invention
The present invention relates to the use of stable HIF-1α variants in accelerating wound healing.
2. Description of the Related Art
HIF-1
Hypoxia-inducible factor-1 (HIF-1) is a mammalian transcription factor that is expressed in response to hypoxia (Wang et al. 1995. Proc. Natl. Acad. Sci. USA 92: 5510-5514). HIF-1 transactivates genes encoding several glucose transporters and glycolytic enzymes, as well as genes increasing tissue perfusion such as vascular endothelial growth factor (VEGF), inducible nitric oxide synthase, and erythropoietin (Semenza, G. 1999. Annual Review Cell and Development Biology 15: 551-78.). HIF-1 is a heterodimeric molecule composed of a labile alpha (HIF-1α) and a constitutive beta (HIF-1β/ARNT aryl hydrocarbon nuclear transporter) subunit. In normoxia (normal oxygen tension), HIF-1α protein is rapidly degraded via ubiquitination and proteasomal digestion. In contrast HIF-1β is stable and equivalently expressed in normoxia and hypoxia. Thus the major regulation of the transcriptional activity of HIF-1 is due to the HIF-1α component.
Structural analysis of HIF-1α has indicated that dimerization requires two domains that have been termed HLH and PAS, while DNA binding is mediated by a basic domain (Semenza et al. 1997. Kid. Int. 51: 553-555). Further, two transactivation domains have been identified in the C-terminal half of HIF-1α (Jiang, et al. 1997. J. Biol. Chem. 272: 19253-19260).
HIF-1α degradation is mediated by an approximately 200-amino acid domain that has been termed the “oxygen-dependent degradation domain” (ODD) (Huang, L., J. Gu, M. Schau, and H. Bunn. 1998. Proc. Natl. Acad. Sci. U.S.A. 95: 7987-92). Cells transfected with cDNA encoding HIF-1α in which the ODD is deleted (HIF-1αΔODD) demonstrate constitutively active HIF-1α protein regardless of oxygen tension (Huang, L., J. Gu, M. Schau, and H. Bunn. 1998. Proc. Natl. Acad. Sci. U.S.A. 95: 7987-92). A number of stable forms of HIF-1α with deletions in the ODD are described in U.S. Pat. No. 6,124,131.
HIF-1α is required for both embryonic development (Ryan, H., J. Lo, and R. Johnson. 1998. EMBO Journal 17: 3005-15) (Iyer, N. et al. 1998. Genes and Development 12: 149-62) and growth of tumor explants (Ryan, H., J. Lo, and R. Johnson. 1998. EMBO Journal 17: 3005-15), which underscores a central role of this molecule in the hypoxic response in vivo. In adult animals HIF-1α is overexpressed in epithelial cancers and high-grade pre-malignant lesions (Zhong, H., et al. 1998. Cancer Research 58: 5280-5284), ischemic cardiac muscle (Lee, S., et al. 2000b. New England Journal of Medicine 342: 626-633), and healing wounds (Elson, D. et al. 2000. Cancer Research 60: 6189-6195).
Wound Healing
Wound healing is a multistep, multicellular process that involves shifting oxygen levels in the wound environment. The initial step of wound healing is characterized by clotting, fibrin formation, and neutrophil infiltration. Neutrophil infiltration provides phagocytic and lysosomal activity. A second step involves macrophage and fibroblast infiltration into the wound thereby facilitating debridement and inflammation. In this second step, enzymes are secreted which digest cellular debris and breakdown intercellular matrices which anchor such cellular debris. Thus, through both phagocytosis and the activity of secreted enzymes, large scale removal of wound debris is effected. Anoxic conditions prevail in the interior of the wound until the completion of angiogenesis; i.e., approximately one week after wounding. Subsequent steps involve fibroblast proliferation; extensive collagen production; capillary formation; and finally myofibroblast contraction leading to compression, disappearance of capillaries, and scar tissue formation.
The process of wound healing begins immediately after formation of a wound and involves numerous cell types and complex interactions between multiple biochemical cascades. Growth factors released in the traumatized area stimulate and promote wound healing, such as by stimulating cell migration into the wound area (chemotaxis), proliferation of epithelial cells, muscle cells, endothelial cells, blood cells and fibroblasts (mitogenesis), formation of new blood vessels (angiogenesis), and matrix formation and remodeling of the affected region including re-epithelization by keratinocytes.
Factors that stimulate or accelerate wound healing will find a variety of uses but are of particular importance in the treatment of patients with chronic wounds which may require daily treatment, represent a constant source of pain to the patient, may lead to life threatening infection and are a significant medical expense. Chronic wounds are those which are slow-healing or which do not heal at all and are common to diabetics, cancer patients and those confined to bed for long periods of time.